Difference between revisions of "Part:BBa K3771039"

Revision as of 21:46, 19 October 2021

PpspA-CS-6xHis

Description

This composite part is a component of the IFN-γ sensing system and was used to express the taurine production enzyme, L-cysteine sulfonic acid synthase (CS).

Biology

The ompA promoter facilitates the constitutive expression of OmpA/OprF. Binding of IFN-γ to the OmpA/OprF chimeric protein induces the response of the phage shock protein (Psp) system, a highly conserved stress response system in enterobacteria. [1] Signal transduction from the outer membrane to the inner membrane activates the pspA promoter, initiating expression of CS-his-tag. CS converts o-phospho-l-serine into L-cysteine sulfonic acid in the taurine synthesis L-cysteine sulfinic acid pathway [2].

Fig.1 Taurine pathways in E. coli

Usage

We ligased the cs fragment and pspA promoter on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid. The his-tag allows for confirmation of CS expression by western blot using the anti-6X his-tag antibody.
Characterization

Fig. 2. Colony PCR confirmation of the construction

Reference

1. Darwin AJ. The phage-shock-protein response. Molecular Microbiology. 2005;57(3):621-628. doi:10.1111/j.1365-2958.2005.04694.xhttps://pubmed.ncbi.nlm.nih.gov/16045608/

2. Joo Y-C, Ko YJ, You SK, et al. Creating a New Pathway in Corynebacterium glutamicum for the Production of Taurine as a Food Additive. Journal of Agricultural and Food Chemistry. 2018;66(51):13454-13463. doi:10.1021/acs.jafc.8b05093https://pubmed.ncbi.nlm.nih.gov/30516051/
Sequence and Features BBa_K3771039 SequenceAndFeatures

@@ Line 12: / Line 12: @@
 <p align="center">Fig.1 Taurine pathways in <i>E. coli</i></p>
    <br><b style="font-size:1.3rem">Usage</b><br>
-   <br>We ligased the NPO-OmpA* fragment and pspA-CS-his-tag on the pSU expression vector and transformed it into DH5alpha to complete construction of the plasmid.  The his-tag allows for confirmation of CS expression by western blot using the anti-6X his-tag antibody.<br>
+   <br>We ligased the <i>cs</i> fragment and <i>pspA</i> promoter on the pSU expression vector and transformed it into DH5α to complete construction of the plasmid.
- <br><b style="font-size:1.3rem">Characterization</b><br>
+The his-tag allows for confirmation of CS expression by western blot using the anti-6X his-tag antibody.<br><b style="font-size:1.3rem">Characterization</b><br>
 <div style="width=100%; display:flex; align-items: center; justify-content: center;">
 <img src="https://2021.igem.org/wiki/images/4/46/T--NCKU_Tainan--colony_csad.jpg" style="width:40%;">